Purification of a triple helix formation with an immobilized obligonucleotide

ABSTRACT

A method for double-stranded DNA purification, by which a solution containing DNA in a mixture with other components is passed over a support on which is covalently coupled an oligonucleotide capable of hybridizing with a specific sequence present on the DNA to form a triple helix.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of application Ser. No. 12/478,418, filed Jun. 4,2009, now allowed, which is a continuation of application Ser. No.11/253,616, filed Oct. 20, 2005, now U.S. Pat. No. 7,622,566, which is acontinuation of application Ser. No. 10/275,071, filed Apr. 7, 2003, nowU.S. Pat. No. 7,038,026, which is the National Stage of InternationalApplication PCT/US01/17122, which is a continuation-in-part of U.S.application Ser. No. 09/580,923, filed May 26, 2000, now U.S. Pat. No.6,319,672, which is a continuation-in-part application of U.S.application Ser. No. 08/860,038, filed Jun. 9, 1997, now U.S. Pat. No.6,287,762, which is the U.S. national stage application of InternationalApplication No. PCT FR95/01468, filed Nov. 8, 1995, the contents ofwhich are relied upon and incorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a new method for DNA purification. Themethod according to the invention enables pharmacologically usabledouble-stranded DNA to be purified rapidly. More especially, thepurification method according to the invention involves a specifichybridization between a sequence of the DNA and an oligonucleotide.

Gene and cell therapy techniques are currently undergoing remarkabledevelopment. However, these techniques entail the possibility ofproducing large amounts of DNA of pharmaceutical purity. In effect, inthese new therapies, the medicament often consists of DNA itself, and itis essential to be able to manufacture it in suitable amounts, toisolate it and to purify it in a manner suited to therapeutic use inman.

In recent years, the feasibility of injection of plasmid DNA for genetherapy or vaccination has been demonstrated by numerous reportsdemonstrating that DNA expression vectors can be taken up by variouscell types and genes encoded by these plasmids can be subsequentlyexpressed (Ledley, 1995 Hum. Gene Ther. 6, 1129).

The genes of interest for gene therapy or vaccination applications mayinclude, for example, tumor suppressor gene, suicide genes, oranti-sense sequences. They can also encode proteins such asalpha-fetoprotein AFP (Morinaga, 1983, Proc. Natl. Acad. Sci. USA, 80,4604), enzymes, hormones, cytokines, growth factors such as FGF(Jouanneau et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 2893) or VEGFB(Olofsson B al., 1996, Proceedings 93, 576), clotting factors such asB-deleted Factor VIII (Truett et al., 1985, DNA 4, 333),apolipoproteins, neurotransmitters, neurotrophic factors, natural orchimeric immunoglobulin. Reporter genes such as lacZ encoding theEscherichia coli β-galactosidase are also used.

Major challenges for using plasmid DNA as a gene delivery vector inhuman are i) the manufacture and ii) the purity of this drug product.Technologies for the production of plasmids vectors with high copynumber in Escherichia coli hosts have been recently developed. Theplasmids currently used are either ColE1-derived plasmids such aspBR322, pUC or pBluescript (Lahijani et al., 1996, Hum. Gene Ther., 7,1971) or pCOR plasmids (Soubrier et al., 1999, Gene Therapy, 6, 1482).

The second concern raised by the use of plasmid DNA as a gene therapyvector is the purity of the plasmid vector itself. Current purificationmethods such as ultracentrifugation in CsCl gradients or chromatographycan be inefficient in removing contaminants such as host genomic DNA andRNA or proteins. Particularly, host genomic DNA whose chemical structureis very close to that of plasmid DNA, is extremely difficult to removeusing classical chromatography. Typical concentrations of up to 0.5 to1% host genomic DNA are found in plasmid preparations obtained byclassical chromatography. Therefore, in order to develop plasmid DNA asa safe vector for human gene therapy, there is a need for purificationtechnologies that will lower the content of host genomic DNA down tomuch lower levels, typically 0.1% or even 0.01% or lower.

The present invention describes a simple and especially effective newmethod for DNA purification. It makes it possible, in particular, toobtain especially high purities with high yields. The method accordingto the invention is based essentially on a specific interaction betweena sequence inserted into the DNA to be purified and an oligonucleotidecomposed of natural or modified bases.

It has recently been shown that some oligonucleotides are capable ofinteracting specifically in the wide groove of the DNA double helix toform triple helices locally, leading to an inhibition of thetranscription of target genes (Hélène et Toulmé, Biochim. Biophys. Acta1049 (1990) 99). These oligonucleotides selectively recognize the DNAdouble helix at oligopurine-oligopyrimidine sequences, that is to say atregions possessing an oligopurine sequence on one strand and anoligopyrimidine sequence on the complementary strand, and form a triplehelix locally thereat. The bases of the third strand (theoligonucleotide) form hydrogen bonds (Hoogsteen or reverse Hoogsteenbonds) with the purines of the Watson-Crick base pairs.

A use of this type of interaction to isolate a plasmid has beendescribed in the prior art. Thus, Ito et al. (PNAS 89 (1992) 495)describe the use of biotinylated oligonucleotides capable of recognizinga particular sequence of a plasmid and of forming a triple helixtherewith. The complexes thus formed are then brought into contact withstreptavidin-coated magnetic beads. Interaction between the biotin andthe streptavidin then enables the plasmid to be isolated by magneticseparation of the beads followed by elution. However, this method hassome drawbacks. In particular, two successive specific interactions areneeded, the first between the oligonucleotide and the plasmid and thesecond between the biotinylated complex and the streptavidin beads.Furthermore, the final solution may be contaminated with biotinylatedoligonucleotide, which cannot be used in a pharmaceutical composition.

SUMMARY OF THE INVENTION

The present invention describes a new, improved method of DNApurification making use of this type of interaction. More especially,the method of the invention employs oligonucleotides coupled covalentlyto a support. This method is especially rapid, and it leads toespecially high yields and degrees of purity. Moreover, it enables DNAto be purified from complex mixtures comprising, in particular, othernucleic acids, proteins, endotoxins (such as lipopolysaccharides),nucleases and the like. The supports used may, in addition, be readilyrecycled, and the DNAs obtained display improved properties ofpharmaceutical safety. Lastly, this method entails only one step,contrary to the prior art.

Hence a first subject of the invention lies in a method for thepurification of double-stranded DNA, according to which a solutioncontaining the said DNA mixed with other components is passed through asupport to which is coupled covalently an oligonucleotide capable offorming a triple helix by hybridization with a specific sequence presentin said DNA. The specific sequence can be a sequence naturally presentin the double-stranded DNA, or a synthetic sequence introducedartificially into the latter.

The oligonucleotides used in the present invention are oligonucleotideswhich hybridize directly with the double-stranded DNA. Theseoligonucleotides can contain the following bases:

-   -   thymidine (T), which is capable of forming triplets with A.T        doublets of double-stranded DNA (Rajagopal et al., Biochem        28 (1989) 7859);    -   adenine (A), which is capable of forming triplets with A.T        doublets of double-stranded DNA;    -   guanine (G), which is capable of forming triplets with G.C        doublets of double-stranded DNA;    -   protonated cytosine (C+), which is capable of forming triplets        with G.C doublets of double-stranded DNA (Rajagopal et al., loc.        cit.);    -   uracil (U), which is capable of forming triplets with A.U or A.T        base pairs.

Preferably, the oligonucleotide used comprises a cytosine-richhomopyrimidine sequence and the specific sequence present in the DNA isa homopurine-homopyrimidine sequence. The presence of cytosines makes itpossible to have a triple helix which is stable at acid pH where thecytosines are protonated, and destablized at alkaline pH where thecytosines are neutralized.

To permit the formation of a triple helix by hybridization, it isimportant for the oligonucleotide and the specific sequence present inthe DNA to be complementary. In this connection, to obtain the bestyields and the best selectivity, an oligonucleotide and a specificsequence which are fully complementary are used in the method of theinvention. These can be, in particular, an oligonucleotide poly(CTT) anda specific sequence poly(GAA). As an example, there may be mentioned theoligonucleotide of sequence 5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (GAGG(CTT)₇;SEQ ID NO: 1), in which the bases GAGG do not form a triple helix butenable the oligonucleotide to be spaced apart from the coupling arm; thesequence (CTT)₇ (SEQ ID No: 26) may also be mentioned. Theseoligonucleotides are capable of forming a triple helix with a specificsequence containing complementary units (GAA). The sequence in questioncan, in particular, be a region containing 7, 14 or 17 GAA units, asdescribed in the examples.

Another sequence of specific interest is the sequence:

5′-AAGGGAGGGAGGAGAGGAA-3′. (SEQ. ID NO: 5)This sequence forms a triple helix with the oligonucleotides

5′-AAGGAGAGGAGGGAGGGAA-3′ (SEQ ID No: 6) or 5′-TTGGTGTGGTGGGTGGGTT-3′.(SEQ ID NO: 7)

In this case, the oligonucleotide binds in an antiparallel orientationto the polypurine strand. These triple helices are stable only in thepresence of Mg²⁺ (Vasquez et al., Biochemistry, 1995, 34, 7243-7251;Beal and Dervan, Science, 1991, 251, 1360-1363).

As stated above, the specific sequence can be a sequence naturallypresent in the double-stranded DNA, or a synthetic sequence introducedartificially in the latter. It is especially advantageous to use anoligonucleotide capable of forming a triple helix with a sequencenaturally present in the double-stranded DNA, for example in the originof replication of a plasmid or in a marker gene. In this connection, theApplicant has performed plasmid sequence analyses, and was able to showthat some regions of these DNAs, in particular in the origin ofreplication, could possess homopurine-homopyrimidine regions. Thesynthesis of oligonucleotides capable of forming triple helices withthese natural homopurine-homopyrimidine regions advantageously enablesthe method of the invention to be applied to unmodified plasmids, inparticular commercial plasmids of the pUC, pBR322, pSV, and the like,type. Among the homopurine-homopyrimidine sequences naturally present ina double-stranded DNA, a sequence comprising all or part of the sequence5′-CTTCCCGAAGGGAGAAAGG-3′ (SEQ ID NO: 2) present in the origin ofreplication of E. coli plasmid ColE1 may be mentioned. In this case, theoligonucleotide forming the triple helix possesses the sequence:5′-GAAGGGCTTCCCTCTTTCC-3′ (SEQ ID NO: 3), and binds alternately to thetwo strands of the double helix, as described by Beal and Dervan (J. Am.Chem. Soc. 1992, 114, 4976-4982) and Jayasena and Johnston (NucleicAcids Res. 1992, 20, 5279-5288). The sequence 5′-GAAAAAGGAAGAG-3′ (SEQID NO: 4) of the plasmid pBR322 β-lactamase gene (Duval-Valentin et al.,Proc. Natl. Acad. Sci. USA, 1992, 89, 504-508) may also be mentioned.

Two additional target sequences which can form triplex structures withparticular oligonucleotides have been identified in ColE1 and in pCORorigins of replication. ColE1-derived plasmids contain a 12-merhomopurine sequence (5′-AGAAAAAAAGGA-3′) (SEQ ID NO: 27) mapped upstreamof the RNA-II transcript involved in plasmid replication (Lacatena etal., 1981, Nature, 294, 623). This sequence forms a stable triplexstructure with the 12-mer complementary 5′-TCTTTTTTTCCT-3′ (SEQ ID NO:28) oligonucleotide. The pCOR backbone contains a homopurine stretch of14 non repetitive bases (5′-AAGAAAAAAAAGAA-3′) (SEQ ID NO: 29) locatedin the A+T-rich segment of the γ origin replicon of pCOR (Levchenko etal., 1996, Nucleic Acids Res., 24, 1936). This sequence forms a stabletriplex structure with the 14-mer complementary oligonucleotide5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30). The corresponding oligonucleotides5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 28) and 5′-TTCTTTTTTTTCTT-3′ (SEQ IDNO:30) efficiently and specifically target their respectivecomplementary sequences located within the origin of replication ofeither ColE1 on or pCOR (oriγ). In fact, a single non-canonical triad(T*GC or C*AT) may result in complete destabilization of the triplexstructure.

The use of an oligonucleotide capable of forming a triple helix with asequence present in an origin of replication or a marker gene isespecially advantageous, since it makes it possible, with the sameoligonucleotide, to purify any DNA containing the said origin ofreplication or said marker gene. Hence it is not necessary to modify theplasmid or the double-stranded DNA in order to incorporate an artificialspecific sequence in it.

Although fully complementary sequences are preferred, it is understood,however, that some mismatches may be tolerated between the sequence ofthe oligonucleotide and the sequence present in the DNA, provided theydo not lead to too great a loss of affinity. The sequence5′-AAAAAAGGGAATAAGGG-3′ (SEQ ID NO: 8) present in the E. coliβ-lactamase gene may be mentioned. In this case, the thymineinterrupting the polypurine sequence may be recognized by a guanine ofthe third strand, thereby forming a G*TA triplet which it is stable whenflanked by two T*AT triplets (Kiessling et al., Biochemistry, 1992, 31,2829-2834).

According to a particular embodiment, the oligonucleotides of theinvention comprise the sequence (CCT)_(n), the sequence (CT)_(n) or thesequence (CTT)_(n), in which n is an integer between 1 and 15 inclusive.It is especially advantageous to use sequences of the type (CT)_(n) or(CTT)_(n). The Applicant showed, in effect, that the purification yieldwas influenced by the amount of C in the oligonucleotide. In particular,as shown in Example 7, the purification yield increases when theoligonucleotide contains fewer cytosines. It is understood that theoligonucleotides of the invention can also combine (CCT), (CT) or (CTT)units.

The oligonucleotide used may be natural (composed of unmodified naturalbases) or chemically modified. In particular, the oligonucleotide mayadvantageously possess certain chemical modifications enabling itsresistance to or its protection against nucleases, or its affinity forthe specific sequence, to be increased.

According to the present invention, oligonucleotide is also understoodto mean any linked succession of nucleosides which has undergone amodification of the skeleton with the aim of making it more resistant tonucleases. Among possible modifications, oligonucleotidephosphorothioates, which are capable of forming triple helices with DNA(Xodo et al., Nucleic Acids Res., 1994, 22, 3322-3330), as well asoligonucleotides possessing formacetal or methylphosphonate skeletons(Matteucci et al., J. Am. Chem. Soc., 1991, 113, 7767-7768), may bementioned. It is also possible to use oligonucleotides synthesized withα anomers of nucleotides, which also form triple helices with DNA (LeDoan et al., Nucleic Acids Res., 1987, 15, 7749-7760). Anothermodification of the skeleton is the phosphoramidate link. For example,the N^(3′)—P^(5′) internucleotide phosphoramidate link described byGryaznov and Chen, which gives oligonucleotides forming especiallystable triple helices with DNA (I Am. Chem. Soc., 1994, 116, 3143-3144),may be mentioned. Among other modifications of the skeleton, the use ofribonucleotides, of 2′-O-methylribose, phosphodiester, etc. (Sun andHélène, Curr. Opinion Struct. Biol., 116, 3143-3144) may also bementioned. Lastly, the phosphorus-based skeleton may be replaced by apolyamide skeleton as in PNAs (peptide nucleic acids), which can alsoform triple helices (Nielsen et al., Science, 1991, 254, 1497-1500; Kimet al., J. Am. Chem. Soc., 1993, 115, 6477-6481), or by aguanidine-based skeleton, as in DNGs (deoxyribonucleic guanidine, Proc.Natl. Acad. Sci. USA, 1995, 92, 6097-6101), or by polycationic analoguesof DNA, which also form triple helices.

The thymine of the third strand may also be replaced by a 5-bromouracil,which increases the affinity of the oligonucleotide for DNA (Povsic andDervan, J. Am. Chem. Soc., 1989, 111, 3059-3061). The third strand mayalso contain unnatural bases, among which there may be mentioned7-deaza-2′-deoxyxanthosine (Milligan et al., Nucleic Acids Res., 1993,21, 327-333),1-(2-deoxy-β-D-ribofuranosyl)-3-methyl-5-amino-1H-pyrazolo[4,3-d]pyrimidin-7-one(Koh and Dervan, J. Am. Chem. Soc., 1992, 114, 1470-1478), 8-oxoadenine,2-aminopurine, 2′-O-methylpseudoisocytidine, or any other modificationknown to a person skilled in the art (for a review see Sun and Hélène,Curr. Opinion Struct. Biol., 1993, 3, 345-356).

Another type of modification of the oligonucleotide has the aim, moreespecially, of improving the interaction and/or affinity between theoligonucleotide and the specific sequence. In particular, a mostadvantageous modification according to the invention consists inmethylating the cytosines of the oligonucleotide (see Example 5). Theoligonucleotide thus methylated displays the noteworthy property offorming a stable triple helix with the specific sequence in pH rangescloser to neutrality (≧5). It hence makes it possible to work at higherpH values than the oligonucleotides of the prior art, that is to say atpH values where the risks of degradation of plasmid DNA are muchsmaller.

The length of the oligonucleotide used in the method of the invention isat least 3 bases, and preferably between 5 and 30. An oligonucleotide oflength greater than 10 bases is advantageously used. The length may beadapted by a person skilled in the art for each individual case to suitthe desired selectivity and stability of the interaction.

The oligonucleotides according to the invention may be synthesized byany known technique. In particular, they may be prepared by means ofnucleic acid synthesizers. Any other method known to a person skilled inthe art may quite obviously be used.

To permit its covalent coupling to the support, the oligonucleotide isgenerally functionalized. Thus, it may be modified by a thiol, amine orcarboxyl terminal group at the 5′ or 3′ position. In particular, theaddition of a thiol, amine or carboxyl group makes it possible, forexample, to couple the oligonucleotide to a support bearing disulphide,maleimide, amine, carboxyl, ester, epoxide, cyanogen bromide or aldehydefunctions. These couplings form by establishment of disulphide,thioether, ester, amide or amine links between the oligonucleotide andthe support. Any other method known to a person skilled in the art maybe used, such as bifunctional coupling reagents, for example.

Moreover, to improve the hybridization with the coupled oligonucleotide,it can be advantageous for the oligonucleotide to contain an “arm” and a“spacer” sequence of bases. The use of an arm makes it possible, ineffect, to bind the oligonucleotide at a chosen distance from thesupport, enabling its conditions of interaction with the DNA to beimproved. The arm advantageously consists of a linear carbon chain,comprising 1 to 18 and preferably 6 or 12 (CH₂) groups, and an aminewhich permits binding to the column. The arm is linked to a phosphate ofthe oligonucleotide or of a “spacer” composed of bases which do notinterfere with the hybridization. Thus, the “spacer” can comprise purinebases. As an example, the “spacer” can comprise the sequence GAGG. Thearm is advantageously composed of a linear carbon chain comprising 6 or12 carbon atoms.

For implementation of the present invention, different types of supportmay be used. These can be functionalized chromatographic supports, inbulk or prepacked in a column, functionalized plastic surfaces orfunctionalized latex beads, magnetic or otherwise. Chromatographicsupports are preferably used. As an example, the chromatographicsupports capable of being used are agarose, acrylamide or dextran aswell as their derivatives (such as Sephadex, Sepharose, Superose, etc.),polymers such as poly(styrene/divinylbenzene), or grafted or ungraftedsilica, for example. The chromatography columns can operate in thediffusion or perfusion mode.

To obtain better purification yields, it is especially advantageous touse, on the plasmid, a sequence containing several positions ofhybridization with the oligonucleotide. The presence of severalhybridization positions promotes, in effect, the interactions betweenthe said sequence and the oligonucleotide, which leads to an improvementin the purification yields. Thus, for an oligonucleotide containing nrepeats of (CCT), (CT) or (CTT) motifs, it is preferable to use a DNAsequence containing at least n complementary motifs, and preferably n+1complementary motifs. A sequence carrying n+1 complementary motifs thusaffords two positions of hybridization with the oligonucleotide.Advantageously, the DNA sequence contains up to 11 hybridizationpositions, that is to say n+10 complementary motifs.

The method according to the present invention can be used to purify anytype of double-stranded DNA. An example of the latter is circular DNA,such as a plasmid, generally carrying one or more genes of therapeuticimportance. This plasmid may also carry an origin of replication, amarker gene, and the like. The method of the invention may be applieddirectly to a cell lysate. In this embodiment, the plasmid, amplified bytransformation followed by cell culture, is purified directly afterlysis of the cells. The method of the invention may also be applied to aclear lysate, that is to say to the supernatant obtained afterneutralization and centrifugation of the cell lysate. It may quiteobviously be applied also to a solution prepurified by known methods.This method also enables linear or circular DNA carrying a sequence ofimportance to be purified from a mixture comprising DNAs of differentsequences. The method according to the invention can also be used forthe purification of double-stranded DNA.

The cell lysate can be a lysate of prokaryotic or eukaryotic cells.

As regards prokaryotic cells, the bacteria E. coli, B. subtilis, S.typhimurium or Strepomyces may be mentioned as examples. As regardseukaryotic cells, animal cells, yeasts, fungi, and the like, may bementioned, and more especially Kluyveromyces or Saccharomyces yeasts orCOS, CHO, C127, NIH3T3, and the like, cells.

The method of the invention is especially advantageous, since it enablesplasmid DNA of very high purity to be obtained rapidly and simply. Inparticular, as illustrated in the examples, this method enables theplasmid DNA in question to be separated effectively from contaminatingcomponents such as fragmented chromosomal DNA, endotoxins, proteins,nucleases, and the like. More especially, the method of the inventionenables preparation of double-stranded DNA, in particular that ofplasmid origin, having a chromosomal DNA content of less than or equalto 0.5% to be obtained. Still more preferably, the DNA preparationsobtained have a chromosomal DNA content of less than or equal to 0.2%.The present invention hence describes compositions comprising plasmidDNA which can be used pharmaceutically, in particular in gene or celltherapy. In this connection, the subject of the invention is also apharmaceutical composition comprising double-stranded DNA, linear or ofplasmid origin, prepared according to the method described above.

The invention also relates to plasmid DNA preparations having achromosomal DNA content of less than or equal to 0.5%, preferably lessthan or equal to 0.2% and still more preferably less than or equal to0.1%, and still more preferably less than or equal to 0.01%. Asexemplified below, a triplex affinity interaction step has beenincorporated in a purification process downstream of classicalchromatographic steps. This affinity step significantly improves thepurity of the plasmid preparation, whatever its initial purity. Theformation of a triplex structure between an oligonucleotide (covalentlybound to a chromatography support) and the plasmid of interest to bepurified relies upon the presence on the plasmid of a sequence that canform a triplex structure with the oligonucleotide. This triplexstructure is stable at acidic pH only, where the cytosines of theoligonucleotide are protonated. Then, plasmid DNA is eluted of thecolumn simply by raising the pH to neutral.

The compositions can contain plasmid DNA which is “naked” or combinedwith transport carriers such as liposomes, nanoparticles, cationiclipids, polymers, recombinant viruses or proteins, and the like.

In one embodiment, the method according to the present invention can beused to purify one type of double-stranded DNA from a mixture comprisingtwo or more double-stranded DNAs of different types and sequences. Thismethod may be applied directly to a cell lysate, in which thedouble-stranded DNAs, amplified through cell culture, are purified afterlysing the cultured cells. This method may also be applied to a clearlysate, i.e., to the supernatant obtained after neutralization andcentrifugation of the cell lysate. The method may further be applied toa prepurified solution.

More precisely, the method for purifying a first double-stranded DNAfrom a solution containing first and second double-stranded DNAs,comprises (i) passing the solution through a first support comprising acovalently coupled oligonucleotide capable of forming a triple helixwith the second double-stranded DNA by hybridization with a specificsequence therein, (ii) recovering the solution that passes through thefirst support, which will be enriched with unbound, firstdouble-stranded DNA, and (iii) passing the recovered solution through asecond support comprising a covalently coupled oligonucleotide capableof forming a triple helix by hybridization with a specific sequence ofsaid first double-stranded DNA. Following an optional washing step, thefirst double-stranded DNA can be eluted from the second support. Usingthis double purification method, the first double-stranded DNA can berecovered from the second support without any detectable levels of thesecond double-stranded DNA.

In a specific embodiment of the present invention, the firstdouble-stranded DNA molecule is a pCOR plasmid having a specificsequence 5′-AAGAAAAAAAAGAA-3′ (SEQ ID NO: 29), which forms a stabletriplex structure with an oligonucleotide having a sequence5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30). The second double-stranded DNAmolecule is a ColE1-derived plasmid having a specific sequence5′-AGAAAAAAAGGA-3′ (SEQ ID NO: 27), which forms a triplex with anoligonucleotide having a sequence 5′TCTTTTTTTCCT-3′ (SEQ ID NO: 28).Accordingly, the pCOR plasmid is advantageously purified from a solutioncontaining other plasmids such as ColE1-derived plasmids by using thedouble purification method according to the present invention.

The present application will be described in greater detail by means ofthe examples which follow, which are to be regarded as illustrative andnon-limiting.

DETAILED DESCRIPTION

General Techniques of Cloning and Molecular Biology

The traditional methods of molecular biology, such as digestion withrestriction enzymes, gel electrophoresis, transformation in E. coli,precipitation of nucleic acids and the like, are described in theliterature (Maniatis et al., T., E. F. Fritsch, and J. Sambrook, 1989.Molecular cloning: a laboratory manual, second edition. Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, New York;Ausubel F. M., R. Brent, R. E. Kinston, D. D. Moore, J. A. Smith, J. G.Seidman and K. Struhl. 1987. Current protocols in molecular biology1987-1988. John Willey and Sons, New York). Nucleotide sequences weredetermined by the chain termination method according to the protocolalready published (Ausubel et al., 1987).

Restriction enzymes were supplied by New England Biolabs, Beverly, Mass.(Biolabs).

To carry out ligations, DNA fragments are incubated in a buffercomprising 50 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 10 mM DTT, 2 mM ATP inthe presence of phage T4 DNA ligase (Biolabs).

Oligonucleotides are synthesized using phosphoramidite chemistry withthe phosphoramidites protected at the β position by a cyanoethyl group(Sinha, N.D., J. Biernat, J. McManus and H. Köster, 1984. Polymersupport oligonucleotide synthesis, XVIII: Use ofβ-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite ofdeoxynucleosides for the synthesis of DNA fragments simplifyingdeprotection and isolation of the final product. Nucl. Acids Res., 12,4539-4557: Giles, J. W. 1985. Advances in automated DNA synthesis. Am.Biotechnol., November/December) with a Biosearch 8600 automatic DNAsynthesizer, using the manufacturer's recommendations.

Ligated DNAs or DNAs to be tested for their efficacy of transformationare used to transform the following strain rendered competent: E. coliDH5α[F/endA1, hsdR17, supE44, thi-1, recA1, gyrA96, relA1Δ(lacZYA-arqF)U169, deoR, Φ80dlac (lacZΔM15)] (for any Col E1 plasmid);or E. coli XAC-pir (for any pCor-derived plasmid).

Minipreparations of plasmid DNA are made according to the protocol ofKlein et al., 1980.

LB culture medium is used for the growth of E. coli strains (Maniatis etal., 1982). Strains are incubated at 37° C. Bacteria are plated out ondishes of LB medium supplemented with suitable antibiotics.

Example 1 1.1. Preparation of the Column

Equipment

The column used is a 1 ml HiTrap column activated with NHS(N-hydroxysuccinimide, Pharmacia) connected to a peristaltic pump(output<1 ml/min. The specific oligonucleotide used possesses an NH₂group at the 5′ end, its sequence is as follows:

5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (SEQ ID NO: 1)The buffers used in this example are the following:

Coupling buffer: 0.2 M NaHCO₃, 0.5 M NaCl, pH 8.3.

Buffer A: 0.5 M ethanolamine, 0.5 M NaCl, pH 8.3.

Buffer B: 0.1 M acetate, 0.5 M NaCl, pH 4.

Method:

The column is washed with 6 ml of 1 mM HCl, and the oligonucleotidediluted in the coupling buffer (50 nmol in 1 ml) is then applied to thecolumn and left for 30 minutes at room temperature. The column is washedthree times in succession with 6 ml of buffer A and then 6 ml of bufferB. The oligonucleotide is thus bound covalently to the column through aCONH link. The column is stored at 4° C. in PBS, 0.1% NaN₃, and may beused at least four times.

1.2. Construction of Plasmids

The following two oligonucleotides were synthesized.

oligonucleotide 4817: (SEQ ID NO: 9)5′-GATCCGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGAAGA AGAA GAAGAAGG-3′oligonucleotide 4818: (SEQ ID NO: 10)5′-AATTCCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTT CTTCTTCTTCG-3′

These oligonucleotides, when hybridized and cloned into a plasmid,introduce a homopurine-homopyrimidine sequence (GAA)₁₇ (SEQ ID NO: 33)into the corresponding plasmid, as described above.

The sequence corresponding to these two hybridized oligonucleotides wascloned at the multiple cloning site of plasmid pBKS+ (Stratagene CloningSystem, La Jolla Calif.), which carries an ampicillin-resistance gene.To this end, the oligonucleotides were hybridized in the followingmanner: one μg of these two oligonucleotides were placed together in 40ml of a final buffer comprising 50 mM Tris-HCl pH 7.4, 10 mM MgCl₂. Thismixture was heated to 95° C. and was then placed at room temperature sothat the temperature would fall slowly. Ten ng of the mixture ofhybridized oligonucleotides were ligated with 200 ng of plasmid pBKS+(Stratagene Cloning System, La Jolla Calif.) digested with BamHI andEcoRI in 30 μl final. After ligation, an aliquot was transformed intoDH5a. The transformation mixtures were plated out on L mediumsupplemented with ampicillin (50 mg/l) and X-gal (20 mg/l). Therecombinant clones should display an absence of blue colouration on thismedium, contrary to the parent plasmid (pBKS+) which permitsα-complementation of fragment ω of E. coli β-galactosidase. Afterminipreparation of plasmid DNA from 6 clones, they all displayed thedisappearance of the PstI site located between the EcoRI and BamHI sitesof pBKS+, and an increase in molecular weight of the 448-bp PvuII bandcontaining the multiple cloning site. One clone was selected and thecorresponding plasmid was designated pXL2563. The cloned sequence wasverified by sequencing using primer −20 (5′-TGACCGGCAGCAAAATG-3′ (SEQ IDNO: 11)) (Viera J. and J. Messing. 1982. The pUC plasmids, an M13 mp7-derived system for insertion mutagenesis and sequencing with syntheticuniversal primers. Gene, 19, 259-268) for plasmid pBKS+(StratageneCloning System, La Jolla Calif.). Plasmid pXL2563 was purified accordingto Wizard Megaprep kit (Promega Corp. Madison, Wis.) according to thesupplier's recommendations. This plasmid DNA preparation was usedthereafter in examples described below.

1.3. Plasmid Purification

Equipment:

Plasmid pXL2563 (described in 1.2) was purified on the HiTrap columncoupled to the oligonucleotide, described in 1.1., from a solution alsocontaining plasmid pBKS+. The buffers used in this purification are thefollowing:

Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5 to 5.

Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA.

Method:

The column is washed with 6 ml of buffer F, and the plasmids (20 μg ofpXL2563 and 20 μg of pBKS+ in 400 μl of buffer F) are applied to thecolumn and incubated for 2 hours at room temperature. The column iswashed with 10 ml of buffer F and elution is then carried out withbuffer E. The plasmids are detected after electrophoresis on 1% agarosegel and ethidium bromide staining. The proportion of the plasmids in thesolution is estimated by measuring their transforming activity on E.coli.Result:Starting from a mixture containing 30% of pXL2563 and 70% of pBKS+, asolution containing 100% of pXL2563 is recovered at the column outlet.The purity, estimated by the OD ratio at 260 and 280 nm, rises from 1.9to 2.5, which indicates that contaminating proteins are removed by thismethod.

Example 2 2.1.—This Example Describes a Plasmid DNA PurificationExperiment

Coupling of the oligonucleotide (5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (SEQ IDNO: 1)) to the column is performed as described in Example 1. For thecoupling, the oligonucleotide is modified at the 5′ end with an aminegroup linked to the phosphate of the spacer by an arm containing 6carbon atoms (Modified oligonucleotide Eurogentec SA, Belgium). PlasmidpXL2563 was purified using the Wizard Megaprep kit (Promega Corp.,Madison, Wis.) according to the supplier's recommendations. The buffersused in this example are the following:

Buffer F: 0-2 M NaCl, 0.2 M acetate, pH 4.5 to 5.

Buffer E: 1 M Tris-HCl pH 9, 0.5 mM EDTA.

The column is washed with 6 ml of buffer F, and 100 μg of plasmidpXL2563 diluted in 400 μl of buffer F are then applied to the column andincubated for 2 hours at room temperature. The column is washed with 10ml of buffer F and elution is then carried out with buffer E. Theplasmid is quantified by measuring optical density at 260 nm.

In this example, binding is carried out in a buffer whose molarity withrespect to NaCl varies from 0 to 2 M (buffer F). The purification yielddecreases when the molarity of NaCl falls. The pH of the binding buffercan vary from 4.5 to 5, the purification yield being better at 4.5. Itis also possible to use another elution buffer of basic pH: elution wasthus carried out with a buffer comprising 50 mM borate, pH 9, 0.5 mMEDTA.

2.2.—Coupling of the Oligonucleotide

(5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (SEQ ID NO: 1) to the column is carriedout as described in Example 1. Plasmid pXL2563 was purified using theWizard Megaprep kit (Promega Corp., Madison, Wis.) according to thesupplier's recommendations. The buffers used in this example are thefollowing:

Buffer F: 0.1 M NaCl, 0.2 M acetate, pH 5.

Buffer E: 1 M Tris-HCl pH 9, 0.5 mM EDTA.

The column is washed with 6 ml of buffer F, and 100 μg of plasmidpXL2563 diluted in 400 μl of buffer F are then applied to the column andincubated for one hour at room temperature. The column is washed with 10ml of buffer F and elution is then carried out with buffer E. Thecontent of genomic or chromosomal E. coli DNA present in the plasmidsamples before and after passage through the oligonucleotide column ismeasured. This genomic DNA is quantified by PCR using primers in the E.coli galK gene. According to the following protocol: The sequence ofthese primers is described by Debouck et al. (Nucleic Acids Res. 1985,13, 1841-1853):

(SEQ ID NO: 24) 5′-CCG AAT TCT GGG GAC CAA AGC AGT TTC-3′ and(SEQ ID NO: 25) 5′-CCA AGC TTC ACT GTT CAC GAC GGG TGT-3′.The reaction medium comprises, in 25 μl of PCR buffer (Promega France,Charbonnières): 1.5 mM MgCl₂; 0.2 mM dXTP (Pharmacia, Orsay); 0.5 μMprimer; 20 U/ml Taq polymerase (Promega). The reaction is performedaccording to the sequence:

5 min at 95° C.

cycles of 10 sec at 95° C.

-   -   30 sec at 60° C.    -   1 min at 78° C.

10 min at 78° C.

The amplified DNA fragment 124 base pairs in length is separated byelectrophoresis on 3% agarose gel in the presence of SybrGreen I(Molecular Probes, Eugene, USA), and then quantified by reference to anUltrapur genomic DNA series from E. coli strain B (Sigma, ref D4889).

There is 1% of chromosomal DNA in the sample applied to the column, and0.2% in the sample purified on the oligonucleotide column.

Example 3 Experiment on Clear Lysate

This example describes plasmid DNA purification from a clear lysate ofbacterial culture, on the so-called “miniprep” scale: 1.5 ml of anovernight culture of DH5α strains containing plasmid pXL2563 arecentrifuged, and the pellet is resuspended in 100 μl of 50 mM glucose,25 mM Tris-HCl, pH 8, 10 mM EDTA. 200 μl of 0.2 M NaOH, 1% SDS areadded, the tubes are inverted to mix, 150 μl of 3 M potassium acetate,pH 5 are then added and the tubes are inverted to mix. Aftercentrifugation, the supernatant is recovered and loaded onto theoligonucleotide column obtained as described in Example 1. Binding,washes and elution are identical to those described in Example 1.Approximately 1 μg of plasmid is recovered from 1.5 ml of culture. Theplasmid obtained, analysed by agarose gel electrophoresis and ethidiumbromide staining, takes the form of a single band of “supercoiled”circular DNA. No trace of high molecular weight (chromosomal) DNA or ofRNA is detectable in the plasmid purified by this method. The ratio ofthe optical densities at 260 and 280 nm is greater than 2.

Example 4 4.1

This example describes a plasmid DNA purification experiment carried outunder the same conditions as Example 3, starting from 20 ml of bacterialculture of DH5α strains containing plasmid pXL2563. The cell pellet istaken up in 1.5 ml of 50 mM glucose, 25 mM Tris-HCl, pH 8, 10 mM EDTA.Lysis is carried out with 2 ml of 0.2 M NaOH, 1% SDS, and neutralizationwith 1.5 ml of 3 M potassium acetate, pH 5. The DNA is then precipitatedwith 3 ml of 2-propanol, and the pellet is taken up in 0.5 ml of 0.2 Msodium acetate, pH 5, 0.1 M NaCl and loaded onto the oligonucleotidecolumn obtained as described in Example 1. Binding, washing of thecolumn and elution are carried out as described in Example 1, except forthe washing buffer, the molarity of which with respect to NaCl is 0.1M.Approximately 16 μg of plasmid DNA are obtained. The plasmid obtained,analysed by agarose gel electrophoresis and ethidium bromide staining,takes the form of a single band of “supercoiled” circular DNA. No traceof high molecular weight (chromosomal) DNA or of RNA is detectable inthe purified plasmid. Digestion of the plasmid with a restriction enzymegives a single band at the expected molecular weight of 3 kilobases. Theprotein concentration in the samples falls from 125 μg/ml in the clearlysate to less than 1 μg/ml in the purified plasmid (Micro-BCA assay,Pierce). The endotoxin concentration, estimated by LAL assay (Biosepra)is divided by a factor of greater than 10 in the purified plasmid,relative to the starting clear lysate.

4.2

The plasmid used contains a cassette containing the cytomegaloviruspromoter, the gene coding for luciferase and thehomopurine-homopyrimidine sequence (GAA)₁₇ (SEQ ID NO: 33) originatingfrom plasmid pXL2563. The strain DH1 (Maniatis et al., 1989) containingthis plasmid is cultured in a 7-liter fermenter. A clear lysate isprepared from 200 grams of cells: the cell pellet is taken up in 2liters of 25 mM Tris, pH 6.8, 50 mM glucose, 10 mM EDTA, to which 2liters of 0.2 M NaOH, 1% SDS, are added. The lysate is neutralized byadding one liter of 3M potassium acetate. After diafiltration, 4 ml ofthis lysate are applied to a 5 ml HiTrap-NHS column coupled to theoligonucleotide of sequence 5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (SEQ ID NO:1), according to the method described in Example 1.1. Washing andelution are carried out as described in Example 1. Approximately 400micrograms of plasmid are recovered. The level of genomic DNA in thissample, measured by the technique described in Example 2.2, is 0.1%.

Example 5 Use of a Modified Oligonucleotide

This example describes the use of an oligonucleotide bearing methylatedcytosines. The sequence of the oligonucleotide used is as follows:

(SEQ ID NO: 12)5′-GAGG^(Me)CTT^(Me)CTT^(Me)CTT^(Me)CTT^(Me)CCT^(Me)CTT^(Me)CTT-3′

This oligonucleotide possesses an NH₂ group at the 5′ end.^(Me)C=5-methylcytosine. This oligonucleotide enables plasmid pXL2563 tobe purified under the conditions of Example 1 with a binding buffer ofpH 5 (the risk of degradation of the plasmid is thereby decreased).

Example 6

In the above examples, the oligonucleotide used is modified at the5′-terminal end with an amine group linked to the phosphate through anarm containing 6 carbon atoms: NH₂—(CH₂)₆. In this example, the aminegroup is linked to the phosphate of the 5′-terminal end through an armcontaining 12 carbon atoms: NH₂—(CH₂)₁₂. Coupling of the oligonucleotideand passage through the column are carried out as described in Example 2with a buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5. This oligonucleotidemakes it possible to have better purification yields: a 53% yield isobtained, whereas, with the oligonucleotide containing 6 carbon atoms,this yield is of the order of 45% under the same conditions.

Example 7

Following the cloning strategy described in Example 1.2, another twoplasmids carrying homopurine-homopyrimidine sequences were constructed:the plasmid pXL2725 which contains the sequence (GGA)₁₆, (SEQ ID NO: 34)and the plasmid pXL2726 which contains the sequence (GA)₂₅ (SEQ ID NO:35).

Example 7.1 Construction of the Plasmids

Plasmids pXL2725 and pXL2726, analogous to plasmid pXL2563, wereconstructed according to the cloning strategy described in Example 1.2,using the following oligonucleotide pairs:

5986: 5′-GATCC(GA)₂₅GGG-3′ (SEQ ID NO: 13) 5987: 5′-AATTCCC(TC)₂₅G-3′(SEQ ID NO: 14) 5981: 5′-GATCC(GGA)₁₇GG-3′ (SEQ ID NO: 15)5982: 5′-AATT(CCT)₁₇CCG-3′ (SEQ ID NO: 16)

The oligonucleotide pair 5986 and 5987 was used to construct plasmidpXL2726 by cloning the oligonucleotides at the BamHI and EcoRI sites ofpBKS+(Stratagene Cloning System, La Jolla Calif.), while theoligonucleotides 5981 and 5982 were used for the construction of plasmidpXL2725. The same experimental conditions as for the construction ofplasmid pXL2563 were used, and only the oligonucleotide pairs werechanged. Similarly, the cloned sequences were verified by sequencing onthe plasmids. This enabled it to be seen that plasmid pXL2725 possessesa modification relative to the expected sequence: instead of thesequence GGA repeated 17 times, there is GGAGA(GGA)₁₅ (SEQ ID NO: 17).

Example 7.2 Preparation of the Columns and Purification

The oligonucleotides forming triple helices with these homopurinesequences were coupled to HiTrap columns according to the techniquedescribed in Example 1.1. The oligonucleotide of sequence5′-AATGCCTCCTCCTCCTCCTCCTCCT-3′ (SEQ ID NO: 18) was used for thepurification of plasmid pXL2725, and the oligonucleotide of sequence5′-AGTGCTCTCTCTCTCTCTCTCTCTCT-3′ (SEQ ID NO: 19) was used for thepurification of plasmid pXL2726.

The two columns thereby obtained enabled the corresponding plasmids tobe purified according to the technique described in Example 2, with thefollowing buffers:

Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5.

Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA.

The yields obtained are 23% and 31% for pXL2725 and pXL2726,respectively.

Example 8

This example illustrates the influence of the length of the specificsequence present in the plasmid on the purification yields.

Example 8.1 Construction of the Plasmids

The reporter gene used in these experiments to demonstrate the activityof the compositions of the invention is the gene coding for luciferase(Luc).

The plasmid pXL2621 contains a cassette containing the 661-bpcytomegalovirus (CMV) promoter, extracted from pcDNA3 (Invitrogen Corp.,San Diego, Calif.) by cleavage with the restriction enzymes MluI andHindIII, cloned upstream of the gene coding for luciferase, at the MluIand HindIII sites, into the vector pGL basic Vector (Promega Corp.,Madison, Wis.). This plasmid was constructed using standard techniquesof molecular biology.

The plasmids pXL2727-1 and pXL2727-2 were constructed in the followingmanner:

Two micrograms of plasmid pXL2621 were linearized with BamHI; the enzymewas inactivated by treatment for 10 min at 65° C.; at the same time, theoligonucleotides 6006 and 6008 were hybridized as described for theconstruction of plasmid pXL2563.

(SEQ ID NO: 20) 6006: 5′-GATCT(GAA)₁₇CTGCAGATCT-3′ (SEQ ID NO: 21)6008: 5′-GATCAGATCTGCAG(TTC)₁₇A-3′.

This hybridization mixture was cloned at the BamHI ends of plasmidpXL2621 and, after transformation into DH5α, recombinant clones wereidentified by PstI enzymatic restriction analysis, since theoligonucleotides introduce a PstI site. Two clones were selected, andthe nucleotide sequence of the cloned fragment was verified using theprimer (6282, 5′-ACAGTCATAAGTGCGGCGACG-3′(SEQ ID NO: 22)) as asequencing reaction primer (Viera J. and J. Messing, 1982. The pUCplasmids an M13 mp 7-derived system for insertion mutagenesis andsequencing with synthetic universal primers. Gene 19:259-268).

The first clone (pXL2727-1) contains the sequence GAA repeated 10 times.The second (pXL2727-2) contains the sequence5′-GAAGAAGAG(GAA)₇GGAAGAGAA-3′ (SEQ ID NO: 23).

Example 8.2 Preparation of the Columns and Purification

A column such as the one described in Example 1, and which is coupled tothe oligonucleotide 5′-GAGGCTTCTTCTTCTTCTTCTTCTT-3′ (SEQ ID NO: 1), isused.

The plasmid pXL2727-1 carries 14 repeats of the sequence GAA. Theoligonucleotide described above, which contains only 7 repeats of thecorresponding hybridization sequence CTT, can hence hybridize with theplasmid at 8 different positions. Plasmid pXL2727-2, in contrast,possesses a hybridizing sequence (GAA)₇ (SEQ ID NO: 36) of the samelength as that of the oligonucleotide bound to the column. Thisoligonucleotide can hence hybridize at only one position on pXL2727-2.

The experiment is identical to the one described in Example 2, with thefollowing buffers:

Buffer F: 2 M NaCl, 0.2 M acetate, pH 4.5.

Buffer E: 1 M Tris-HCl, pH 9, 0.5 mM EDTA.

The purification yield is 29% with plasmid pXL2727-1 and 19% withpXL2727-2.

Example 8.3 In Vitro Transfection of Mammalian Cells

The cells used are NTH 3T3 cells, inoculated on the day before theexperiment into 24-well culture plates on the basis of 50,000cells/well. The plasmid is diluted in 150 mM NaCl and mixed with thelipofectant RPR115335. A lipofectant positive charges/DNA negativecharges ratio equal to 6 is used. The mixture is vortexed, left for tenminutes at room temperature, diluted in medium without foetal calf serumand then added to the cells in the proportion of 1 μg of DNA per culturewell. After two hours at 37° C., 10% volume/volume of foetal calf serumis added and the cells are incubated for 48 hours at 37° C. in thepresence of 5% of CO2. The cells are washed twice with PBS and theluciferase activity is measured according to the protocol described(Promega kit, Promega Corp. Madison, Wis.) on a Lumat LB9501 luminometer(EG and G Berthold, Evry). Plasmid pXL2727-1, purified as described inExample 8.2, gives transfection yields twice as large as those obtainedwith the same plasmid purified using the Wizard Megaprep kit (PromegaCorp. Madison, Wis.).

Example 9 Purification of pCOR-Derived Plasmids

The following example demonstrates the purification of pCOR-derivedplasmids using triple-helix affinity chromatography. This technology hasbeen shown to remove nucleic acid contaminants (particularly hostgenomic DNA and RNA) down to levels that have not been achieved withconventional chromatography methods:

A triplex affinity gel was synthesized with Sephacryl S-1000 SF(Amersham-Pharmacia Biotech) as the chromatography matrix. SephacrylS-1000 was first activated with sodium m-periodate (3 mM, roomtemperature, 1 h) in 0.2 M sodium acetate (pH 4.7). Then theoligonucleotide was coupled through its 5′-NH₂ terminal moiety toaldehyde groups of the activated matrix by reductive amination in thepresence of ascorbic acid (5 mM) as described previously for thecoupling of proteins (Hornsey et al., J. Immunol. Methods, 1986, 93,83-88). The homopyrimidine oligonucleotide used for these experiments(from Eurogentec, HPLC-purified) had a sequence which was complementaryto a short 14-mer homopurine sequence (5′-AAGAAAAAAAAGAA-3′) (SEQ ID NO:29) present in the origin of replication (oriγ) of the pCOR plasmid(Soubrier et al., Gene Therapy, 1999, 6, 1482-1488). As discussed above,the sequence of the homopyrimidine oligonucleotide is5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30).

The following plasmids were chromatographed: pXL3296 (pCOR with notransgene, 2.0 kpb), pXL3179 (pCOR-FGF, 2.4 kpb), pXL3579 (pCOR-VEGFB,2.5 kbp), pXL3678 (pCOR-AFP, 3.7 kbp), pXL3227 (pCOR-lacZ 5.4 kbp) andpXL3397 (pCOR-Bdeleted FVIII, 6.6 kbp). All these plasmids were purifiedby two anion-exchange chromatography steps from clear lysates obtainedas described in example 4. Plasmid pBKS+(pBluescript II KS+ fromStratagene), a ColE1-derived plasmid, purified by ultracentrifugation inCsCl was also studied. All plasmids used were in their supercoiled(>95%) topological state.

In each plasmid DNA purification experiment, 300 μg of plasmid DNA in 6ml of 2 M NaCl, 0.2 M potassium acetate (pH 5.0) was loaded at a flowrate of 30 cm/h on an affinity column containing the above-mentionedoligonucleotide 5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30). After washing thecolumn with 5 volumes of the same buffer, bound plasmid was eluted with1 M Tris/HCl, 0.5 mM EDTA (pH 9.0) and quantitated by UV (260 nm) andion-exchange chromatography with a Millipore Gen-Pak column (Marquet etal., BioPharm, 1995, 8, 26-37). Plasmid recoveries in the fractioncollected were 207 μg for pXL3296, 196 μg for pXL13179, 192 μg forpXL3579, 139 μg for pXL3678, 97 μg for pXL3227, and 79 μg for pXL 3397.

No plasmid binding could be detected (<3 μg) when pBKS waschromatographed onto this column. This indicates that oligonucleotide5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30) makes stable triplex structureswith the complementary 14-mer sequence 5′-AAGAAAAAAAAGAA-3′ (SEQ ID NO:29) present in pCOR (oriγ), but not with the closely related sequence5′-AGAAAAAAAGGA-3′ (SEQ ID NO: 27) present in pBKS. This indicates thatthe introduction of a single non-canonical triad (T*GC in this case)results in a complete destabilization of the triplex structure.

As a control, no plasmid binding (<1 μg) was observed when pXL3179 waschromatographed on a blank column synthesized under strictly similarconditions but without oligonucleotide.

By operating this affinity purification column in the conditionsreported here, the level of contamination by host genomic DNA wasreduced from 2.6% down to 0.07% for a preparation of pXL3296. Similarlythe level of contamination by host DNA was reduced from 0.5% down to0.008% for a preparation of pXL3179 when the sample was chromatographedthrough the same affinity column. In addition, the level ofcontamination by RNA was largely reduced from 43% RNA down to 0.2% RNAin a preparation of pXL3179 by using this affinity purification column.

In addition, plasmid PXL3579 recovery was less than 8% whenoligonucleotide 5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30) was replaced byoligonucleotide 5′-TTTTTTTTCTT-3′ (SEQ ID NO: 31) on the affinitycolumn. While the oligonucleotide as set forth in SEQ ID NO: 31 iscomplementary to a portion of the VEGFB sequence within pXL3579 (i.e.,nucleotides 379 to 389 relative to ATG), no significant triplex affinityoccurs. This indicates that this affinity purification requires anon-random homopurine-homopyrimidine DNA sequence.

Example 10 Purification of a ColE1-Derived Plasmid

The following example demonstrates the purification of ColE1-derivedplasmids using triple-helix affinity chromatography. This technology hasbeen shown to remove nucleic acid contaminants (particularly hostgenomic DNA and RNA) down to levels that have not been achieved withconventional chromatography methods.

A triplex affinity gel was synthesized by coupling of an oligonucleotidehaving the sequence 5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 28) ontoperiodate-oxidized Sephacryl S-1000 SF as described in Example 9.

Plasmids pXL3296 (pCOR with no transgene) and pBKS, a ColE1-derivedplasmid, were chromatographed on a 1-ml column containingoligonucleotide 5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 28) in conditionsdescribed in Example 9. Plasmid recoveries in the fraction collectedwere 175 μg for pBKS and <1 μg for pXL3296. This indicates thatoligonucleotide 5′-TCTTTTTTTCCT-3′ (SEQ ID NO: 28) makes stable triplexstructures with the complementary 12-mer sequence (5′AGAAAAAAAGGA-3′)(SEQ ID NO: 27) present in pBKS, but not with the very closely related12-mer sequence (5′-AGAAAAAAAAGA-3′) (SEQ ID NO: 32) present in pCOR.This indicates that the introduction of a single non-canonical triad(C*AT in this case) may result in complete destabilization of thetriplex structure.

Example 11 Double Purification Method

The following example demonstrates the purification of a supercoileddouble-stranded DNA molecule, such as pXL3296, in a mixture containinganother supercoiled double-stranded molecule, such as pBSK, using triplehelix affinity chromatography. Both double-stranded DNA molecules mayhave a similar size, but each DNA molecule contains a unique sequencethat is capable of forming a triple helix with a different targetsequence. As previously discussed, molecules such as pXL3296 contain asequence 5′-AAGAAAAAAAAGAA-3′ (SEQ ID NO: 29), but do not contain thesequence 5′-AGAAAAAAAGGA-3′ (SEQ ID NO: 27). In contrast, molecules suchas pBSK contain SEQ ID NO: 27, but do not contain SEQ ID NO: 29.

In a first step, the mixture containing pXL3296 and pBSK was loaded on afirst affinity column containing the oligonucleotide 5′-TCTTTTTTTCCTT-3′(SEQ ID NO: 28), such as the column described in Example 10. Thesolution was passed through the first column which contained unbound DNAmolecules. In the second step, the unbound DNA molecules from the firststep were loaded on a second affinity column containing theoligonucleotide 5′-TTCTTTTTTTTCTT-3′ (SEQ ID NO: 30), such as the columndescribed in Example 9. The second column was then washed and the boundmolecules were eluted, as described in Example 9. Only pXL3296 moleculeseluted from the second column. No pBSK molecules were detected in theeluate (i.e., the solution that elutes from the column) from the secondcolumn.

1. A triple helix bound to a purification support that is afunctionalized chromatographic support, wherein the triple helixcomprises a pyrimidine-rich oligonucleotide covalently coupled directlyto the support through an amide or amine link and a double-stranded DNAthat has hybridized to the oligonucleotide.
 2. The triple helixaccording to claim 1, wherein the oligonucleotide comprises acytosine-rich sequence.
 3. The triple helix according to claim 1,wherein the oligonucleotide comprises a thymidine-rich sequence.
 4. Thetriple helix according to claim 1, wherein the oligonucleotide compriseseither a first sequence (CCT)_(n), a second sequence (CT)_(n), a thirdsequence (CTT)_(n), or a combination of any of the first, second, andthird sequences, wherein n is an integer between 1 and 15 inclusive. 5.The triple helix according to claim 1, wherein the support furthercomprises an arm comprising a carbon chain (CH₂)_(n), wherein n is aninteger between 1 and 18 inclusive, and wherein the arm is linked to theoligonucleotide through a phosphate and to the support through an amidelink.
 6. The triple helix according to claim 5, wherein n is an integerbetween 6 and
 12. 7. A method of making a support for purifyingdouble-stranded DNA, comprising covalently coupling an oligonucleotideto a functionalized chromatographic support, wherein the oligonucleotidecomprises a pyrimidine-rich sequence, and forming a triple helix with adouble-stranded DNA by hybridizing the oligonucleotide with a nucleotidesequence in the double-stranded DNA, and wherein the oligonucleotide iscovalently coupled directly to the support through a disulphide,thioether, ester, amide or amine link.
 8. The method according to claim7, wherein the oligonucleotide comprises a cytosine-rich sequence. 9.The method according to claim 7, wherein the oligonucleotide comprises athymidine-rich sequence.
 10. The method according to claim 7, whereinthe oligonucleotide comprises either a first sequence (CCT)_(n), asecond sequence (CT)_(n), a third sequence (CTT)_(n), or a combinationof any of the first, second, and third sequences, wherein n is aninteger between 1 and 15 inclusive.
 11. The method according to claim 7,wherein the oligonucleotide is coupled to the support through an amideor amine link.
 12. The method according to claim 7, wherein theoligonucleotide is coupled to the support via an arm comprising a carbonchain (CH₂)_(n), wherein n is an integer between 1 and 18 inclusive, andwherein the arm is linked to the oligonucleotide through a phosphate andto the support through an amide link.
 13. The method according to claim12, wherein n is an integer between 6 and
 12. 14. The support obtainedby the method of claim
 7. 15. A method of coupling an oligonucleotide toa purification support, wherein the support is a functionalizedchromatographic support that comprises residues, comprising: a)activating the residues of the support, and b) contacting the activatedresidues of the support with the oligonucleotide to obtain covalentcoupling directly between the oligonucleotide and the residues of thepurification support; wherein the oligonucleotide comprises apyrimidine-rich sequence which forms a triple helix with adouble-stranded DNA by hybridizing with a nucleotide sequence in thedouble-stranded DNA.
 16. A functionalized chromatographic supportcomprising a covalently coupled oligonucleotide, wherein theoligonucleotide comprises a pyrimidine-rich sequence which forms atriple helix with a double-stranded DNA by hybridizing with a nucleotidesequence in the double-stranded DNA, wherein the oligonucleotide iscovalently coupled directly to the purification support through adisulphide link.